The present invention relates to a chimeric molecule comprising an oligonucleotide complementary to some portion of the ribonucleotide component of telomerase and an activator of RNase L (xe2x80x9cactivator-antisense complexesxe2x80x9d). The present invention relates to compounds useful for the treatment of disorders and diseases related to enhanced or elevated telomerase activity. In particular, the present invention relates to compounds and methods of their use for treating humans having a malignant neoplastic disease of the type, wherein the malignant cells contain a telomerase enzyme that is necessary for the continued growth of the tumor. Particularly, the invention concerns a RNase L activator antisense complex that targets the RNA component of telomerase enzymes, cleaves the RNA and inhibits telomerase activity. More particularly, the invention relates to activator-antisense complexes, in which the oligonucleotide is selected to bind to regions of the ribonucleotide component of telomerase that possess repeated or consensus sequences. The invention further relates to activator-antisense complexes, in which the oligonucleotide is selected to bind to a portion of the RNA component that normally has no self-hybridizing secondary structure.
2.1 ACTIVATOR-ANTISENSE COMPLEXES
Activator-antisense complexes (termed therein xe2x80x9c2-5A:ASxe2x80x9d) have been described previously for use to cleave specifically selected strands of RNA (Torrence et al., 1993, WO 94/09129 by Torrence et al., U.S. Pat. No. 5,583,032). The mechanism of action of activator-antisense complexes is different than the mechanism of action of other antisense oligonucleotides. The activator portion of the activator-antisense complexes activates RNase L and the antisense domain serves as a specific, high affinity binding site for the target RNA. The result is the selective cleavage of the target RNA by RNase L.
Physiologically, RNase L functions as part of the interferon system in restricting virus replication in cells of higher vertebrates (reviewed in Silverman, 1994). Interferon treatment of cells activates genes encoding 2-5A synthetases, double-stranded RNA (dsRNA)-dependent enzymes that produce 5xe2x80x2-triphosphorylated, 2xe2x80x2,5xe2x80x2-linked oligoadenylates (2xe2x80x2,5xe2x80x2A) from ATP. Viral dsRNAs are potential activators of these enzymes (Gribaudo et al., 1991, J. Virol. 65, 1748). The 2xe2x80x2,5xe2x80x2A binds to and activates RNase L resulting in the general cleavage of cellular and viral RNA; thus restricting the replication of some picornaviruses (Chebath et al., 1987, Nature 330, 587; Rysiecki et al., 1989, J. Interferon Res. 9, 649; and Hassel et al., 1994, EMBO J. 12, 3297).
RNase L is not specific for cleaving viral RNA. For instance, in interferon-treated, encephalomyocarditis virus infected cells, RNase L causes degradation of ribosomal RNA (Wreschner et al., 1981, Nucleic Acid Res. 9, 1571). Through the activator-antisense approach, RNase L is converted from a non-specific nuclease to a highly specific endoribonuclease that selectively cleaves mRNA targets. This has been demonstrated in a cell-free system from Daudi cells, a human lymphoblastoid cell line, in which a modified HIV-1 vif mRNA was targeted for cleavage by an activator-antisense complex (Torrence et al., 1993, Proc. Natl. Acad. Sci. USA 90, 1300). Subsequently, purified RNase L has been directed by an activator-antisense complex to cleave selectively an mRNA target encoding the protein kinase PKR in the presence of a nontargeted mRNA (Maran et al., 1994, Science 265, 789). Furthermore, in HeLa cells, the use of activator-antisense complexes, which were directed to a sequence in PKR mRNA, resulted in the ablation of PKR mRNA and enzyme activity (ibid.) such that the dsRNA-mediated activation of transcription factor, NF-kB was ablated. More recently, it was shown that the activation of RNase L by an activator-antisense complex results in the catalytic degradation of PKR mRNA (kcat of about 7 secxe2x88x921) (Maitra et al., 1995, J. Biol. Chem. 270, 15071).
2.2 TELOMERASE
Telomeres correspond to the ends of eukaryotic chromosomes and are specialized structures containing unique (TTAGGG)N repeats. (Blackburn, 1991, Nature 350, 569-573). Telomeres protect the chromosomes from DNA degradation, end-to-end fusions, rearrangements, and chromosome loss. (deLange, T., 1994, Proc. Natl. Acad. Sci. 91, 2882-85). Because cellular DNA polymerases cannot replicate the 5xe2x80x2 end of the linear DNA molecules found in eukaryotic chromosomes, the number of telomere repeats decreases by 50-200 nucleotides/cell division during aging of normal somatic cells. (Harley, et al., 1990, Nature 345, 458-460; Hastie, et al., 1990, Nature 346, 866-68). Shortening of telomeres may also control the proliferative capacity of normal cells. Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5xe2x80x2-TTAGGG-3xe2x80x2 to the end of telomeres to prevent progressive loss. (Greider et al., 1985, Cell 43, 405-413). Although the vast majority of normal somatic cells do not express telomerase, most types of tumor cells express telomerase at high levels. (Kim et al., 1994, Science 266, 2011-15; Broccoli et al., 1995, Proc. Natl. Acad. Sci. 92, 9082-86; Hiyama et al., 1995, J. Natl. Cancer Inst. 87, 895-902; Hiyama et al., 1995, Cancer Res. 55, 3258-62). High levels of telomerase in a tumor are correlated with a poor prognosis thought to be necessary for a cell to become malignant and conversely low levels of telomerase are associated with a favorable prognosis. (Hiyama, supra). Although telomerase-independent mechanisms for telomere maintenance cannot be formally excluded, (Rogan et al., 1995, Mol. Cell. Biol. 15, 4745-53; Strahl et al., 1996, Mol. Cell. Biol. 16, 53-652), telomerase activity is most likely to be the dominant mechanism. (Holt et al., 1996, Mol. Cell. Biol. 16, 2932-39). Thus, telomerase activity is considered to be a necessary factor for the malignant transformation of a cell.
Recently, it has been observed that when HeLa cells were transfected with an plasmid that expresses an mRNA complementary to the RNA component of human telomerase, the transfected cells were found to lose telomeric DNA and to die after 23 to 26 doublings. (Feng et al., 1995, Science 269, 1236-41).
U.S. Pat. No. 5,583,016 Villeponteau (xe2x80x9cVilleponteauxe2x80x9d) discloses the sequence of the RNA component of human telomerase (xe2x80x9chTRxe2x80x9d). Villeponteau describes in theory the use of oligonucleotides for the inhibition of telomerase gene expression or oligonucleotides which bind to the RNA component of telomerase and prevent it from acting as a substrate in humans. U.S. Pat. Nos. 5,489,508 and 5,645,986 to West describe both therapeutics and diagnostic assays directed to telomerase, in particular, oligonucleotides which could be designed to bind to a telomerase RNA component, prevent telomerase from binding to a telomere and thereby inhibit telomerase activity. These U.S. patents also describe the use of antisense oligonucleotides to inhibit telomerase gene expression. Those groups have described in theory, methods of inhibiting telemerase activity by targeting the ability of telomerase to extend telomeres or by inhibiting telomerase gene expression.
The present invention relates to chimeric molecules comprising an oligonucleotide complementary to a region of the ribonucleotide component of telomerase attached to an activator of RNase L (xe2x80x9cactivator-antisense complexxe2x80x9d) which specifically cleaves the ribonucleotide portion of a telomerase enzyme. The present invention relates to methods of inhibiting telomerase enzymatic activity with activator-antisense complexes targeted to the RNA component of telomerase.
The present invention provides a complex that is useful for the treatment of malignant disease by inhibition of telomerase activity. The essential components of the complex are an antisense oligonucleotide which has a sequence that is complementary to between about 12 and about 25 nucleotides of the RNA component of human telomerase and an activator of RNase L (henceforth, an xe2x80x9canti-telomerase activator-antisense complexxe2x80x9d). In a preferred embodiment, the antisense portion of the activator-antisense complex is complementary to 18 or 19 nucleotides of the RNA component of telomerase. The elements of the activator-antisense complex may be covalently or non-covalently linked.
The activator-antisense complexes of the present invention can be transported across the cell membrane without the use of carriers or permeabilizing agents. The activator-antisense complexes of the present invention can also be used with an agent to facilitate transport across a cell membrane, e.g. lipofectamine. Once internalized the activator-antisense complexes lead to the formation of enzyme-antisense complexes, which causes destruction of the telomerase RNA.
The invention is based, in part, on the Applicants"" unexpected result that exposure of a human malignant glioma cell to a 2-5A-activator linked to 19-mer antisense oligonucleotide targeted to the RNA component of telomerase (2-5A-hTR) effectively suppressed tumor cell growth and survival in vitro and in vivo. As shown by the Applicants, the dose of 2-5A-hTR required to block proliferation and survival of tumor cells can be reduced by 10-fold using lipofectamine to facilitate cellular uptake of the oligonucleotide. Applicants have found that malignant glioma cells treated with 2-5A-anti-hTR undergo cell death by apoptosis. Applicants have also demonstrated that when 2-5A-anti-hTR was administered in vivo to mice with intracranial implantations of human malignant gliomes, 50% of the mice survived at 62 days post-tumor implantation. The Applicants have further demonstrated that a broad range of cancer cell types are susceptible to the antiproliferative effects of 2-5A-anti-hTR.
The present invention further relates to pharmaceutical compositions comprising the anti-telomerase activator-antisense complexes for the treatment and prevention of cell proliferative disorders related to enhanced telomerase activity, e.g., malignant neoplastic disease. The present invention also relates to methods of treating and preventing cell proliferative disorders, such as cancer, related to enhanced telomerase activity including, but not limited to, brain cancer, breast cancer, prostate cancer, renal cancer and melanoma. In particular, the present invention relates to methods of treating and preventing the growth of tumors, wherein the tumor cells contain a telomerase enzyme that is necessary for the continued growth of the tumor.